The TSG101 tumor susceptibility gene initially was identified by the reversible neoplasia associated with deficiency of its protein product in mouse fibroblasts (1). Deficiency of TSG101 induced by antisense RNA in NIH3T3 cells leads to colony formation in 0.5% agar, focus formation in monolayer cell cultures, and the ability to form metastatic tumors in athymic nude mice (1). Turn off of TSG101-inactivating antisense RNA reverses these features of neoplastic transformation as well as the nuclear, microtubule, and mitotic spindle abnormalities observed in TSG101-deficient cells (1, 2). The steady-state level of TSG101 protein normally is regulated post-translationally in cells within a narrow range (3), and overexpression of TSG101 from an adventitious promoter can also lead to cell cycling abnormalities (2) and neoplastic transformation (1). Truncated TSG101 transcripts, which are observed in a variety of human tumors as well as in normal cells (4-7), have been attributed to aberrant or alternative RNA splicing (8) and have been correlated with both cellular stress (4, 5) and mutation of p53 (5). The TSG101 protein contains motifs common to transcription regulators (1) and can modulate transcriptional activation by steroid hormone receptors (9-11).
Sequence analysis has also suggested that TSG101, which is expressed in mammalian cells from the earliest stages of embryonic development and in multiple tissues of adult mice (8), may additionally have a role in the regulation of ubiquitin-mediated proteolysis (12, 13). The N-terminal region of the TSG101 protein contains a domain (Ubc) that resembles the catalytically active region of ubiquitin conjugases (E2 enzymes) but lacks an active site cysteine residue crucial to the function of these enzymes (12-14), leading to speculation that TSG101 may act as a dominant negative inhibitor of ubiquitination (12, 13).
p53 is a key tumor suppressor that transcriptionally activates MDM2 as well as other genes implicated in both cell growth and cell death (15-18). MDM2 in turn negatively regulates p53 by promoting its ubiquitin-mediated degradation (19-21). Despite p53/MDM2 feedback control, p53 accumulates in cells soon after DNA damage, hypoxia, and other types of stress, suggesting that the actions of MDM2 and p53 on each other are themselves regulated (16). Several mechanisms for such regulation have been proposed (16, 17) and recent evidence indicates that alteration of MDM2 stability mediated by its interactions with other cellular proteins may have a role in this process (22-24).
There is a continuing need in the art for new and better methods of modulating proliferation of mammalian cells. The present invention addresses this need.